Metabolism of 26,27-hexafluoro-1α,25-dihydroxyvitamin D3 and 26,27-hexafluoro1α,23(S)25-trihydroxyvitamin D3 in ROS17/2.8 cells transfected with a plasmid expressing CYP24

1. To clarify the possibility that the metabolism of 26,27-hexafl uoro-1α, 25-dihydroxyvitamin D₃ [F₆-1,25(OH)₂D₃] to 26,27-hexafluoro-1α,23(S),25-trihydroxyvitamin D₃ [F₆-1,23,25(OH)₃D₃ and that of F₆-1,23,25(OH)₃D₃ to 26,27-hexafluoro-23-oxo-1α,25-dihydroxyvitamin D₃ [F₆-23-oxo-1,25(OH)₂D₃] are catalysed by 25-hydroxyvitamin D 24-hydroxylase (CYP24), ROS17}2.8 cells transfected with a plasmid expressing CYP24 [pSVL-CYP24–] and a corresponding blank plasmid [pSLVCYP24R(®)] were used. 2. Incubation of [1β-³H]-F-1,25(OH)₂D₃ for 2 and 5 days with ROS17}2.8 cells transfected with pSVL-CYP24– generated a metabolite that co-migrated with authentic F₆ -1,23,25(OH)₃D₃ in both normal phase and reversed-phase HPLC systems. 3. Incubation of [1β-³H]-F₆ -1,23,25(OH)₃D₃ for 5 days with pSVL-CYP24– - transfected ROS 17}2.8 cells generated a metabolite that co-migrated with authentic F₆ -23-oxo-1,25(OH)₂D₃. In contrast, the metabolites F₆ -1,23,25(OH)₃D₃ or F₆ -23-oxo- 1,25(OH)₂D₃ were not generated in the cells transfected with pSVL-CYP24R(-). 4. The results indicate that CYP24 catalyses the conversion of F₆ -1,25(OH)₂D₃ to F₆ -1,23,25(OH)₃D₃ and that of F₆ -1,23,25(OH)₃D₃ to F₆ -23-oxo-1,25(OH)₂D₃.     

Metabolism of 26,26,26,27,27,27-F6-1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3). The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3). The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3). In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24. Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway. Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.     

Metabolism of 26,26,26,27,27,27-F6-1 alpha,23S,25-trihydroxyvitamin D3 by human UDP-glucuronosyltransferase 1A3.

26,26,26,27,27,27-Hexafluoro-1alpha,25-dihydroxyvitamin D(3) [F(6)-1alpha, 25(OH)(2)D(3)], which is now clinically used as a drug for secondary hyperparathyroidism, is a hexafluorinated analog of the active form of vitamin D(3). Our previous studies demonstrated that CYP24A1 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues and that F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3). In this study, we examined the metabolism of F(6)-1alpha,25(OH)(2)D(3),F(6)-1alpha,23S,25(OH)(3)D(3), and F(6)-23-oxo-1alpha,25(OH)(2)D(3) by human UDP-glucuronosyltransferases (UGTs). Of these compounds, F(6)-1alpha,23S,25(OH)(3)D(3) was remarkably glucuronidated both in human liver microsomes and in the recombinant system expressing human UGT. No significant interindividual differences were observed among 10 human liver samples. The recombinant system for 12 species of human UGTs revealed that F(6)-1alpha,23S,25(OH)(3)D(3) glucuronidation was specifically catalyzed by UGT1A3. The information obtained in this study seems very useful to predict the metabolism and efficacy of vitamin D analogs in human bodies before clinical trials. In addition, note that for the first time a possible probe substrate for UGT1A3 has been found.     

Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1alpha,25-dihydroxyvitamin D(3): implications for drug-induced osteomalacia

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3). No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)(2)D(3) hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)(3)D(3), 1,24S,25(OH)(3)D(3), and 1,23S,25(OH)(3)D(3). Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)(2)D(3) 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)(2)D(3) metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.     

Disposition and metabolism of F6-1alpha,25(OH)2 vitamin D3 and 1alpha,25(OH)2 vitamin D3 in the parathyroid glands of rats dosed with tritium-labeled compounds.

26,26,26,27,27,27-Hexafluoro-1alpha,25(OH)2 vitamin D3, a hexafluorinated analog of 1alpha,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound with respect to some vitamin D actions. The reason for enhanced biological activity in the bones, kidneys, and small intestine appears to be related to F6-1alpha,25(OH)2 vitamin D3 metabolism to ST-232 (26,26,26,27,27,27-hexafluoro-1alpha,23S,25-trihydroxyvitamin D3), a bioactive 23S-hydroxylated form that is resistant to further metabolism. We compared the disposition and metabolism of [1beta-3H]F6-1alpha,25(OH)2 vitamin D3 and [1beta-3H]1alpha,25(OH)2 vitamin D3 in parathyroid glands of rats intravenously administered with labeled compounds at a dose of 10 microg/kg. In the [1beta-3H]F6-1alpha,25(OH)2 vitamin D3-dosed group, radioactivity was highly detected in the kidneys, parathyroid glands, and the small intestine. The radioactivity in the parathyroid glands remained high until 48 h postdosing, with values of 2.5, 8.4, and 14.6 times higher at 6, 24, and 48 h postdosing than after dosing with [1beta-3H] 1alpha,25(OH)2 vitamin D3. In the group given [1beta-3H]F6-1alpha,25(OH)2 vitamin D3, the unchanged compound was mainly detected with a small amount of ST-232 at 6 h postdosing. At the 24- and 48-h time points, over half of the radioactivity was observed as ST-232, and additionally, ST-233, the 23-oxo form, accounted for a small amount at the 48-h time point. The present study demonstrated local retention of [1beta-3H]F6-1alpha,25(OH)2 vitamin D3 and the bioactive metabolite ST-232 in parathyroid glands after intravenous administration. The findings may indicate one of the reasons for the higher potency of F6-1alpha,25(OH)2 vitamin D3 than 1alpha,25(OH)2 vitamin D3 in parathyroid.     

In vivo and in vitro pharmacokinetics and metabolism studies of 26,26,26,27,27,27-F6-1,25(OH)2 vitamin D3 (Falecalcitriol) in rat: induction of vitamin D3-24-hydroxylase (CYP24) responsible for 23S-hydroxylation in target tissues and the drop in serum levels.

1. 26,26,26,27,27,27-F6,-1,25(OH)2 vitamin D3, Falecalcitriol, the hexafluorinated analogue of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biological activity appears related to F6-1,25(OH)2 vitamin D3 metabolism to F6-1,23S,25(OH)3 vitamin D3, a bioactive 23S-hydroxylated form which is resistant to further metabolism. 2. In the present in vivo studies, the repeated oral administration of [3H]F6-1,25(OH)2 vitamin D3 to rat resulted in a significant reduction of the radioactivity and the F6-1,25(OH)2 vitamin D3 concentrations in serum, especially at the 2 h maximum point after each dosing. Additionally, F6-1,23S,25(OH)3 vitamin D3 in the serum and small intestine was increased by the prior administration of F6-1,25(OH)2 vitamin D3. 3. Further in vitro investigation showed [3H]F6-1,25(OH)2 vitamin D3 to be metabolized to F6-1,23S,25(OH)3 vitamin D3 by kidney and small intestine homogenates of rat, the reaction being increased by the prior administration of F6-1,25(OH)2 vitamin D3. Moreover, this latter treatment was associated with a marked increase of CYP24 mRNA in the small intestine within 4 h after dosing. 4. The results indicate that in vivo metabolism of F6-1,25(OH)2 vitamin D3 to F6-1,23S,25(OH)3 vitamin D3 is catalysed by CYP24, the enzyme being induced by prior substrate exposure.     

Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells.

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1 alpha,25-(OH)2D3 > or = 1 alpha,25-(OH)2-26,27-CH3)2D3 > 1 alpha,25-(OH)2-26,27-(C2H5)2D3 > 1 alpha,25-(OH)2-26,27-(C3H7)2D3. These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1 alpha,25-(OH)2D3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1 alpha-OH group from 1 alpha,25-(OH)2D3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1 alpha-OH groups in the 1 alpha,25-(OH)2D3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1 alpha,25-(OH)2D3 receptor of HL-60 cells, suggesting a 1 alpha,25-(OH)2D3 receptor-mediated induction mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biological activity of novel vitamin D analogues: 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 and 24,24-difluoro-1 alpha,25-dihydroxy-26,27-dimethylvitamin D3

We synthesized 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 (16), and 24,24-difluoro-1 alpha, 25-dihydroxy-26,27-dimethylvitamin D3 (21), from 3 beta-hydroxy-22,23-dinorcholenic acid 3-acetate. Compound 16 was found to be a highly potent vitamin D analogue with bioactivity similar to that of 25-hydroxyvitamin D3 in vivo. Compound 16 was bound by vitamin D binding protein with an affinity slightly less than that of 25-hydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with approximately the same affinity as that of 25-hydroxyvitamin D3. In the organ-culture duodenum, 16 induced the synthesis of calcium binding protein with a potency approximately 1/20 that of 1,25-dihydroxyvitamin D3. Compound 21 was also noted to be a highly potent vitamin D analogue with bioactivity in vivo similar to that of 1,25-dihydroxyvitamin D3. It was bound to vitamin D binding protein with an affinity considerably less than that of 1,25-dihydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with an affinity slightly less than that of the native hormone. In the organ-culture duodenum, 21 was noted to be about 3 times more active than 1,25-dihydroxyvitamin D3 in the induction of calcium binding protein. The introduction of fluorines at C-24 and extension of the sterol side chain at C-26 and C-27 by methylene groups results in vitamin D analogues that have biological activity in vivo similar to those of the respective nonfluorinated natural sterols.     

In vitro biological activities of a series of 2 beta-substituted analogues of 1 alpha,25-dihydroxyvitamin D3

Biological activities of a series of 2beta-substituted analogues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] were evaluated in vitro in terms of their binding affinity with regard to calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). Additionally, reporter gene luciferase activities using either a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), in transfected rat osteoblast-like ROS17/2.8 cells, or a human VDR-GAL4 modified two-hybrid system in transfected human epitheloid carcinoma, cervix HeLa cells were examined. Binding affinity for VDR, transactivation potency on the target gene and VDR-mediated gene regulation of the hydroxyalkyl and hydroxyalkoxy 2beta-substituted analogues were almost comparable to those of 1alpha,25(OH)2D3, while the alkyl and alkenyl analogues were much less active than 1alpha,25(OH)2D3. This study investigated the biological evaluation of a series of 2beta-substituted analogues at the molecular level, with regard to the structural differences of alkyl, alkenyl, hydroxyalkyl, hydroxyalkoxy, alkoxy, hydroxy and chloro substituents at the 2beta-position of 1alpha,25(OH)2D3.     

Isolation and identification of 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3: metabolites of 1alpha,24(R)-dihydroxyvitamin D3 produced in rat kidney

1alpha,24(R)-Dihydroxyvitamin D3 [1alpha,24(R)(OH)2D3], a synthetic vitamin D3 analog, has been developed as a drug for topical use in the treatment of psoriasis. At present, the target tissue metabolism of 1alpha,24(R)(OH)2D3 is not understood completely. In our present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in the isolated perfused rat kidney. The results indicated that 1alpha,24(R)(OH)2D3 is metabolized in rat kidney into several metabolites, of which 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxovitamin D3, 1alpha,23(S),25-trihydroxy-24-oxovitamin D3, and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D3 are similar to the previously known metabolites of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. In addition to these aforementioned metabolites, we also identified two new metabolites, namely 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3. The two new metabolites do not possess the C-25 hydroxyl group. Thus, the metabolism of 1alpha,24(R)(OH)2D3 into both 25-hydroxylated and non-25-hydroxylated metabolites suggests that 1alpha,24(R)(OH)2D3 is metabolized in the rat kidney through two pathways. The first pathway is initiated by C-25 hydroxylation and proceeds further via the C-24 oxidation pathway. The second pathway directly proceeds via the C-24 oxidation pathway without prior hydroxylation at the C-25 position. Furthermore, we demonstrated that rat kidney did not convert 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] into 1alpha,25(OH)2D3. This finding indicates that the rat kidney does not possess the classical vitamin D3-25-hydroxylase (CYP27) activity. However, from our present study it is apparent that prior hydroxylation of 1alpha(OH)D3 at the C-24 position in the 'R' orientation allows 25-hydroxylation to occur. At present, the enzyme responsible for the C-25 hydroxylation of 1alpha,24(R)(OH)2D3 is unknown. Our observation that the side chain of 1alpha,24(R)(OH)2D3 underwent 24-ketonization and 23-hydroxylation even in the absence of the C-25 hydroxyl group suggests that 1alpha,25(OH)2D3-24-hydroxylase (CYP24) can perform some steps of the C-24 oxidation pathway without prior C-25 hydroxylation. Thus, we speculate that CYP24 may be playing a dual role in the metabolism of 1alpha,24(R)(OH)2D3.     

Kinetic studies of 25-hydroxy-19-nor-vitamin D3 and 1 alpha,25-dihydroxy-19-nor-vitamin D3 hydroxylation by CYP27B1 and CYP24A1.

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D(3) [25(OH)-19-nor-D(3)] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)], suggesting that 25(OH)-19-nor-D(3) might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D(3) [1 alpha,25(OH)(2)-19-nor-D(3)] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) and 25-hydroxyvitamin D(3) [25(OH)D(3)] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3), we found that k(cat)/K(m) for 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) was less than 0.1% of that for 25(OH)D(3), and the k(cat)/K(m) value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)(2)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3). The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D(3) compared with 1 alpha,25(OH)(2)D(3). We then analyzed the metabolites of 25(OH)D(3) and 25(OH)-19-nor-D(3) in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha,25(OH)(2)D(3) was detected in cells incubated with 25(OH)D(3), whereas no 1 alpha,25(OH)(2)-19-nor-D(3) was detected in cells incubated with 25(OH)-19-nor-D(3). Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D(3) is converted to 1 alpha,25(OH)(2)-19-nor-D(3) by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D(3) by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D(3) has potential as an attractive agent for prostate cancer therapy.     

Metabolism of 2 alpha-propoxy-1 alpha,25-dihydroxyvitamin D3 and 2 alpha-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 by human CYP27A1 and CYP24A1.

Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.     

Preliminary study on serum levels of 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D in cadmium-induced renal tubular dysfunction

Serum 1,25-dihydroxyvitamin D [1,25(OH)2D] and 25-hydroxyvitamin D [25(OH)D] were measured in 7 women living in a cadmium-polluted area in Toyama, Japan. Despite the fact that these subjects had severe proximal renal tubular dysfunction showing increased fractional excretion of beta 2-microglobulin (FE beta 2-m) ranging from 9.7-49.1% with a mean of 30.7%, the levels of serum 1,25(OH)2D, which is produced in the proximal tubules, were within the normal range in 6 subjects. Significant correlations were found between 1,25(OH)2D and creatinine clearance (r = 0.802, P less than 0.05), and FE beta 2-m (r = -0.829, P less than 0.05), respectively. These results suggest that renal production of 1,25(OH)2D decreases with progression of cadmium-induced renal tubular dysfunction.     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Rapid control of transmembrane calcium influx by 1alpha,25-dihydroxyvitamin D3 and its analogues in rat osteoblast-like cells.

1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3] has been shown to exert both its nuclear vitamin D receptor (nVDR)-mediated genomic actions and membrane vitamin D receptor (mVDR)-mediated nongenomic actions. In this study, the effects of 1alpha,25(OH)2D3 and its analogues on transmembrane Ca2+ influx were examined in the growth phase of rat osteosarcoma ROS17/2.8 cells. Like BAYK8644 (2 x 10(-5)M), a well-known L-type Ca2+ channel agonist, 1alpha,25(OH)2D3 (10(-8)M) increased transmembrane influx of Ca2+ through voltage-dependent Ca2+ channels and increased intracellular Ca2+ concentration within 2 min of addition to the medium. The 1alpha,25(OH)2D3-induced Ca2+ influx was completely blocked by pre-treatment with nifedipine (2 x 10(-5)M), an L-type Ca2+ channel antagonist. Two vitamin D analogues, 22-oxa-1alpha,25(OH)2D3 (OCT, 10(-8) M) and 20-epi-22-oxa-24a, 26a,27a-trihomo-1alpha,25(OH)2D3 (KH1060, 10(-8)M), which were 3.8 and 3600-fold more active than 1alpha,25(OH)2D3 in stimulating differentiation on human promyelocytic leukemic HL-60 cells, respectively, also increased intracellular Ca2+ concentration, while their Ca2+ channeling activities were similar to or significantly weaker than that of 1alpha,25(OH)2D3. Furthermore, the enhanced transmembrane Ca2+ influx induced by 1alpha,25(OH)2D3 (10(-8)M) or OCT (10(-8)M) was completely blocked by pre-treatment with the respective 1beta epimer [1beta,25(OH)2D3 and 1beta-OCT] at equal concentration. These findings suggest that 1alpha,25(OH)2D3 and its analogues modulate transmembrane Ca2+ influx in osteoblast-like cells by opening L-type Ca2+ channels which can recognize 1alpha-hydroxy analogues as agonists and 1beta-hydroxy analogues as antagonists.